PROJECT SUMMARY Intracellular cargo transport is a vital and ubiquitous cellular process that occurs in all eukaryotic cells. However, the mechanisms of cargo transport have only been extensively investigated in a small number of ?model? species, leaving a large knowledge gap in our understanding of cargo transport in other eukaryotic groups. My laboratory has recently uncovered a novel mechanism of cargo transport in the Apicomplexan protozoan parasite Toxoplasma gondii; elucidating the mechanistic details underlying this intracellular transport system is the focus on this proposal. T. gondii is part of the phylum Apicomplexan, which contains a large number of medically important parasites including Plasmodium spp., the causative agent of malaria and Cryptosporidium spp. that can cause life-threatening diarrheal disease. Thus, in addition to providing insight into the diverse mechanisms of cargo transport that exist in evolutionary divergent eukaryotes, understanding the molecular basis of cargo transport in Apicomplexa could lead to the identification of potential targets of anti-parasitic drug development. Our previously published and preliminary data demonstrate that two cytoskeletal proteins, actin and an unconventional myosin (MyoF) are required for the movement of a wide range of intracellular cargo. Although MyoF has structural similarity to the well-characterized cargo transporter myosin V, we demonstrate that MyoF does not associate directly with its membrane bound cargo. Instead, MyoF is an organizer of the underlying actin cytoskeleton. In order to gain a complete picture of this cargo transport mechanism, our goal is to identify additional molecular players that are required for cargo transport and to define the mechanisms by which these proteins drive cargo movement in collaboration with MyoF and actin. Specifically, we will answer the following outstanding questions: How is the activity of MyoF regulated? How does vesicular cargo associate with the actin cytoskeleton? Are there additional proteins that cooperate with MyoF to control the organization and dynamics of the actin cytoskeleton? To address these questions, we will utilize three distinct, yet complementary, experimental approaches: (1) parasite genetics and cell biology, (2) live cell imaging and quantitative vesicle tracking and (3) in vitro biophysical approaches. My laboratory occupies a unique niche at the intersection between parasitology and molecular motors fields. Utilizing these interdisciplinary approaches makes us ideally positioned to provide new insights into this understudied process.